[unreadable] [unreadable] Transfer RNA (tRNA) plays a crucial role in the expression of genetic information in all living organisms. In addition to being an essential component of the cellular translation machinery, tRNA also plays a role in priming RNA-directed DNA synthesis of several retroviral genomes, including HIV. Thus, elucidating the mechanisms of tRNA formation and function has important implications in both physiological and pathological gene expression. Newly synthesized pre-tRNAs undergo extensive nuclear processing to become competent for nucleocytoplasmic transport and protein synthesis. A general feature of tRNA molecules is the presence of a vast array of modified nucleosides formed by numerous enzymes, yet the role of many of these nucleoside derivatives in tRNA metabolism and function is poorly understood. One of these modifications, 1-methyladenosine (m1A), and the m1A methyltransferase (Mtase) are the focus of this proposal. Unlike other tRNA modification enzymes, the yeast m1A Mtase has two subunits, encoded by GCD10 and GCD14, and each of these genes is essential for cell viability. Furthermore, homologs of these genes exist in a variety of eukaryotic organisms. These observations underscore the importance of m1A formation in cellular physiology. The long-term goals of this project are to uncover the significance of the two-subunit structure of this enzyme and to understand how m1A formation contributes to the regulation of tRNA function and gene expression. [unreadable] The enzymology of the m1A Mtase will be studied using combination of biochemical and molecular biology methods to express and purify yeast or recombinant enzyme complex as well as individual protein subunits. These will be examined in vitro in terms of tRNA recognition and binding, and the role of each subunit in the reaction will be defined with regard to substrate and methyl donor binding. As the first essential tRNA modification identified in yeast, new genetic strategies can be employed to complement in vitro studies by generating and screening mutant alleles of GCD10 and GCD14 for loss of function in vivo. The effect of these mutations on enzyme properties will be ascertained to define protein determinants involved in subunit, substrate or cofactor binding or methyltransferase activity. In addition to understanding how the m1A Mtase modifies specific tRNA substrates, alternative RNA substrates will be identified and characterized to further probe the cellular role(s) of GCD10 and GCD14. These studies will provide important new information in the area of tRNA metabolism and have the potential for introducing novel concepts in the field of post-transcriptional control of gene expression that may be broadly applicable to higher eukaryotic systems. [unreadable] [unreadable] [unreadable]